Brief Report
Azithromycin Inhibits Macrophage Tumor Necrosis Factor Secretion in Response to Both AzithromycinSusceptible and Azithromycin-Resistant Pneumococci Kim Ingram,1,2 Matthew Marker,1 Elizabeth Meals,1 Ajay J. Talati,1,3 Thomas Spentzas,1,2,5 and B. Keith English1,4 1
Children’s Foundation Research Institute at Le Bonheur Children’s Hospital; Divisions of 2Pediatric Critical Care Medicine, Neonatology, and 4Pediatric Infectious Diseases, Department of Pediatrics, and 5Department of Preventive Medicine, University of Tennessee Health Science Center, Memphis
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Corresponding Author: Kimberly Ingram, MD, 50 N Dunlap, Rm 408R Memphis, TN 38103. E-mail: ingram.kimberly@ gmail.com. Received September 4, 2012; accepted January 29, 2013; electronically published April 5, 2013.
We studied the effect of azithromycin (AZM) on macrophage responses to pneumococci. We found that exposure of pneumococci to AZM led to reduced tumor necrosis factor (TNF) secretion by macrophages; this effect was observed in response to both AZM-susceptible and AZM-resistant (AZM-R) pneumococci. Key words.
ampicillin; azithromycin; macrophage; pneumococci; TNF.
Streptococcus pneumoniae is the leading cause of bacterial pneumonia in children and adults, and penicillin and other beta-lactam agents are recommended as first-line treatment for this condition [1]. Although resistance to beta-lactam antibiotics rarely results in treatment failure in patients with pneumococcal pneumonia, several lines of evidence suggest that beta-lactam monotherapy is not optimal therapy for pneumococcal pneumonia even when the bacteria are fully susceptible [2–6]. Three clinical trials in adult patients with pneumococcal pneumonia suggest that combination therapy (usually with a beta-lactam plus a macrolide) is superior to monotherapy with a betalactam [2–4]. Furthermore, in a mouse model of post-influenzal pneumococcal pneumonia, therapy with macrolide antibiotics such as AZM or lincosamides such as clindamycin was superior to therapy with ampicillin [5]. In that mouse model, the addition of AZM to ampicillin also led to reduced mortality in mice infected with a laboratoryderived, AZM-R pneumococcal strain [6]. Compared with beta-lactam antibiotics, nonlytic antibiotics such as macrolides trigger reduced release of proinflammatory bacterial products, and this may result in a blunted host inflammatory response [5–7]. Azithromycin also reportedly has anti-inflammatory effects unrelated to its antimicrobial activity (eg, in patients with cystic fibrosis [8]).
We hypothesized that exposure of a panel of strains of S pneumoniae (including strains resistant to AZM) to AZM (alone or in combination with ampicillin) compared with ampicillin alone would lead to reduced secretion of TNF by macrophages stimulated with these bacteria.
METHODS Macrophages
RAW 264.7 cells were purchased from the American Type Culture Collection and were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Mediatech Inc., Herndon, VA). Experiments were performed in 24-well tissue culture plates (Becton Dickinson, Lincoln Park, NJ) and the cells were seeded at a concentration of 1 × 106 cells/mL well. Antibiotics
Ampicillin (AMP), azithromycin (AZM), cefotaxime, and clindamycin were obtained from the LeBonheur Children’s Hospital pharmacy (Memphis, TN). Clinically achievable concentrations of antibiotics were used: 20 µg/mL AMP; 20 µg/mL cefotaxime; 10 µg/mL clindamycin; and 1 µg/mL, 5 µg/mL, and 20 µg/mL AZM. Bacteria
Two laboratory strains of Streptococcus pneumoniae were obtained from Dr Jon McCullers (St Jude Children’s
Journal of the Pediatric Infectious Diseases Society, Vol. 3, No. 2, pp. 168–71, 2014. DOI:10.1093/jpids/pit014 © The Author 2013. Published by Oxford University Press on behalf of the Pediatric Infectious Diseases Society. All rights reserved. For Permissions, please e-mail:
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Azithromycin Inhibits Macrophage Tumor Necrosis Factor Secretion in Response to Both Azithromycin-Susceptible
Research Hospital, Memphis, TN) and have been described previously [5, 6] (a luciferase-expressing version of the AZM-susceptible [AZM-S] strain, A66.1 [AZM minimal inhibitory concentration, MIC, 0.25 µg/mL] and a laboratory-derived, AZM-resistant [AZM-R] subclone of A66.1, AZM-R-A66.1 [AZM MIC >256 µg/mL]). We also obtained 4 AZM-R pneumococcal isolates from the Le Bonheur Children’s Hospital microbiology laboratory (AZM-R isolates C1, C2, C3, and C4). We studied 2 isolates with moderately elevated MICs (MIC of C1 was 8 µg/mL; MIC of C2 was 24 µg/mL) and 2 with MICs greater than 256 µg/mL (C3, C4). Bacteria were grown at 37°C in DIFCO Todd Hewitt broth to concentrations of 105–107 at an optical density wavelength of 620 nm. The bacteria were washed 3 times in cold phosphate-buffered saline, and concentrations were determined via colony counts. All strains were susceptible to AMP (MICs were 0.5 µg/mL). Macrophage Stimulation
RAW 264.7 macrophages were exposed to live S pneumoniae bacteria at a range of concentrations (105, 106, or 107 colony-forming units [cfu]/mL), immediately after the addition of antibiotics to the culture medium, and incubated for 18 h. Supernatants were harvested and assayed for tumor necrosis factor (TNF) concentrations using a solid-phase sandwich enzyme-linked immunosorbent assay as specified by the manufacturer (eBioscience Inc., San Diego, CA). Statistical Analysis
The results were analyzed with R 2.13.1 and the ggplot2 graph package. Analysis was validated with SPSS19 (IBM). The experimental design consisted of factorial multiple measurements. The normality was assessed with the Kolmogorov-Smirnov test and plot. The analyses of variance are between independent groups, and we tested the homogeneity of the variance with Levene’s test. We set preplanned (a priori) contrasts, ie, we set all of our comparisons in advance of multiple setting experiments. Significance was presumed at P < .05. When post hoc tests were used we applied the Bonferroni correction. The results were graphed as fold of change compared with the control using error bars with 95% confidence intervals. RESULTS We first studied the A66.1 reference strain of pneumococcus, which is fully susceptible to AMP and AZM. We stimulated RAW 264.7 macrophages with 106 cfu/mL of live A66.1 in the presence of either AMP alone (20 µg/mL) or AZM alone (1, 5, or 20 µg/mL). Macrophages exposed to A66.1 in the presence of AZM (versus AMP) secreted 28%–48% less TNF, and the effect of AZM was
dose-dependent (Figure 1A, top panel). Likewise, addition of AZM to AMP resulted in dose-dependent reductions of 29%–53% in macrophage TNF secretion (Figure 1A, bottom panel). In addition, we found that adding AZM (20 µg/mL) to cefotaxime (20 µg/mL) or clindamycin (10 µg/mL) led to 23%–30% reductions in macrophage TNF secretion after stimulation with A66.1 (data not shown). We next examined a previously characterized [6], laboratory-derived, AZM-R subclone of A66.1 (AZM-RA66.1) (MIC >256 µg/mL). We compared the macrophage TNF response to AZM-R-A66.1 in the presence of either AMP alone (20 µg/mL) or AMP plus AZM (1, 5, or 20 µg/mL) (Figure 1B). As with the AZM-S parental strain, addition of AZM to AMP resulted in dose-dependent reductions in macrophage TNF secretion in response to the AZM-RA66.1 subclone, although the difference was statistically significant only at the higher concentrations of AZM and the magnitude of the reduction (14%–21%) was less than half of that observed in response to AZM-S A66.1 (Figure 1B). To confirm our findings in this laboratory-derived AZM-R pneumococcal strain, we exposed macrophages to each of the 4 AZM-R clinical pneumococcal isolates in the presence of AMP alone or AMP plus AZM (1, 5, or 20 µg/mL). For each of the 4 isolates, addition of AZM to AMP resulted in reductions in macrophage TNF secretion in at least 1 inoculum (Figure 1C depicts the results at inoculum of 106 cfu/mL). Although the magnitude of the reductions after addition of AZM varied, it was statistically significant for 3 of the 4 clinical isolates studied, including 1 of the 2 highly resistant isolates (C3). Exposure to the combination of AMP and AZM decreased TNF production 16%–18% from the control in response to isolate C1, 15%–19% in response to isolate C2, 45%–62% in response to isolate C3, and by only 2% (not significant) in response to strain C4. Finally, we tested the ability of AZM to inhibit macrophage TNF responses to heat-killed pneumococci. Figure 1D depicts the results from experiments using heat-killed AZM-R-A66.1 (results with other pneumococcal strains were similar; data not shown). Incubation with AMP alone had no effect on RAW 264.7 cell TNF secretion in response to heat-killed AZM-R-A66.1, but exposure to AZM alone resulted in a 12%–21% reduction in TNF secretion, whereas addition of AZM to AMP led to a similar (15%–18%) reduction in TNF secretion (P < .05 for the 5 and 20 µg/mL concentrations of AZM).
DISCUSSION We found that exposure of both AZM-S and AZM-R pneumococci to AZM led to decreased macrophage TNF
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Figure 1. Exposure to azithromycin (AZM) leads to reduced tumor necrosis factor (TNF) secretion by RAW 264.7 macrophages exposed to AZM-susceptible (AZM-S) and AZM-resistant (AZM-R) pneumococci. Macrophages (1 × 106 cells/mL) were stimulated overnight with bacteria (106 colony-forming units [cfu]/mL) in the presence of the indicated antibiotics, and cell supernatants were collected and assayed for TNF concentrations. Results are expressed as a percentage reduction of the control (ampicillin [AMP] alone). A, Macrophages secreted less TNF in response to the AZM-S A66.1 strain of Streptococcus pneumoniae in the presence of AZM (alone or in combination with AMP) than in the presence of AMP alone. Exposure to AZM alone (top) compared with AMP led to a dose-dependent decrease in TNF secretion by macrophages stimulated with A66.1. The addition of AZM (bottom) to AMP, compared with AMP alone, led to a dose-dependent reduction in TNF secretion of similar magnitude to that observed in the presence of AZM alone. Statistical significance (P < .05) is designated by an asterisk. B, Macrophages secreted less TNF in response to the AZM-R subclone of pneumococcal strain A66.1 (AZM-R-A66.1) in the presence of AZM plus AMP (compared with AMP alone). The addition of AZM led to dose-dependent reductions in TNF secretion by macrophages in response to this AZM-R strain, although the difference was only statistically significant at higher AZM concentrations. The magnitude of the reduction in TNF secretion was much less than that observed in response to the AZM-S A66.1 parental strain. Statistical significance (P < .05) is designated by an asterisk. C, Macrophages secreted less TNF in response to each of the 4 AZM-R clinical isolates when stimulated in the presence of AZM plus AMP (compared with AMP alone), and this was statistically significant for 3 of the 4 strains. Statistical significance (P < .05) is designated by an asterisk. D, Macrophages secreted less TNF in response to heat-killed pneumococci (AZM-R-A66.1) when stimulated in the presence of AZM (alone or in combination with AMP) compared with AMP alone. Exposure to AZM alone (top) compared with AMP led to a dose-dependent decrease in TNF secretion by macrophages stimulated with the heat-killed bacteria. The addition of AZM to AMP (bottom), compared with AMP alone, led to a dose-dependent reduction in TNF secretion of similar magnitude to that observed in the presence of AZM alone. Statistical significance (P < .05) is designated by an asterisk.
Azithromycin Inhibits Macrophage Tumor Necrosis Factor Secretion in Response to Both Azithromycin-Susceptible
secretion in response to these bacteria. Our findings may have clinical relevance in the context of serious or lifethreatening pneumococcal infections, including sepsis, meningitis, and pneumonia [2-4, 7], and may provide an explanation for the observation that AZM treatment (alone or in combination) of postinfluenzal pneumococcal pneumonia in a mouse model was superior to therapy with AMP [5, 6]. However, the magnitude of reduction of macrophage TNF secretion resulting from the addition of AZM varied and was less marked in response to AZM-R pneumococci. Furthermore, our data cannot determine what magnitude of reduction would be required to result in clinical benefit to a patient with pneumococcal pneumonia. In the mouse model of postinfluenzal pneumococcal pneumonia, little TNF is detected in the serum of the mice [9], but large amounts of TNF and other cytokines are detected in lung homogenates [5, 9]. Mice treated with clindamycin accumulated less than half as much TNF in their lungs compared with mice treated with AMP, but reductions in other lung cytokine concentrations were less dramatic (20%–35%) [5]. The effect of AZM on macrophage TNF secretion was more marked for AZM-S strains and varied significantly amongst the macrolide-resistant clinical isolates. Furthermore, AZM also diminished macrophage TNF responses to heat-killed whole bacteria. Taken together, we interpret these results to indicate that AZM-mediated inhibition of macrophage TNF responses to pneumococci involves both its antimicrobial activity and its nonspecific anti-inflammatory activity. We have previously reported that exposure of pneumococci [10] and other Gram-positive cocci (including group B streptococci) [11] to nonlytic antibiotics (including clindamycin and rifampin) leads to blunted macrophage inflammatory mediator production in response to these pathogens. In addition to the studies of pneumococcal pneumonia cited above, other work in experimental models of serious pneumococcal infections also suggests that treatment with nonlytic antibiotics that trigger less marked macrophage inflammatory responses may be beneficial [7]. Our data provide additional evidence that treatment of serious infections caused by pneumococci and other Gram-positive pathogens with rapidly lytic cell-wall active antibiotics such as beta-lactams may be suboptimal. Additional studies of the efficacy of AZM and other nonlytic antibiotics in the treatment of pneumococcal infections in animal models and clinical trials are needed.
Acknowledgments We thank Dr Jon McCullers for providing the A66.1 strain and the laboratory-derived, azithromycin-resistant subclone of A66.1 and for helpful suggestions. Financial support. This work was supported by a Le Bonheur Children’s Hospital Research Grant (to K. I.). Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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